JPS5912135B2 - enzyme electrode - Google Patents

Description

[Detailed description of the invention] The present invention relates to improvements in enzyme electrodes.

More specifically, it is an electrode for electrochemically measuring the concentration of various substrates, which are substances that undergo the specific catalytic action of enzymes, or converts the chemical energy of the substrate into electrical energy by combining it with a counter electrode such as an enzyme electrode. This invention relates to electrodes used in batteries. Oxidoreductases are mainly involved in energy conversion in living organisms, and there have recently been many attempts to utilize this type of enzymatic reaction industrially.
For example, there are many proposals for so-called enzyme electrodes that combine enzymes with electrochemical measurements to measure the concentration of enzyme substrates. First, regarding the oxidoreductase used in the present invention, the relationship between the oxidation-reduction reaction catalyzed by the oxidoreductase and electrochemical measurement will be explained.

Generally, when a substrate is redoxed by an oxidoreductase, an electron carrier is required.

For example, the enzyme glucose oxidase (hereinafter GOX)
When the oxidation reaction of glucose is carried out using (abbreviated as ), oxygen O2 acts as an electron carrier, particularly an electron acceptor, as shown in the reaction formula below. Glucose + O2 Once → Gluconolactone + H2O2・・・・・・
(1) That is, glucose is oxidized (dehydrogenated) to gluconolactone by the catalytic action of GOX, and at the same time, O2 accepts electrons (hydrogen) from glucose and is reduced to H and O2.

Here, unlike glucose, oxygen or hydrogen peroxide can be used as a target for electrochemical concentration measurement, so the glucose concentration can be indirectly measured from the electrochemical measurement of 02 consumed or H2O2 produced in the above reaction formula (1). Measurement becomes possible. On the other hand, in the case of GOX, the natural electron acceptor 0
2 can be replaced with various artificial electron acceptors collectively known as redox compounds, such as quinone, methylene blue, 2,6-dichlorophenolindophenol, and potassium ferricyanide. For example, when quinone is used in place of 02, the following reaction occurs. Glucose + quinone GOX? Gluconolactone + Hydroquinone (2) Here, since quinone and hydroquinone are electrode active substances and their concentration can be measured electrochemically, it is also possible to measure the concentration of glucose.

In addition to GOX, xanthine oxidase, amino acid oxidase, aldehyde oxidase, etc. are oxidoreductases that can substitute for natural electron acceptors such as 02 and exhibit their effects even with artificial redox compounds. Furthermore, some types of oxidoreductases require one of a group of compounds called coenzymes as electron carriers during redox reactions of substrates.

For example, alcohol dehydrogenase and lactate dehydrogenase require nicotinamide adenine dinucleotide l' (NAD) as a coenzyme. NAD is a typical coenzyme, but other coenzymes such as nicotinamide adenine dinucleotide phosphate, pyridoxal phosphate, coenzyme A, lipoic acid, and folic acid are also known as coenzymes. It is practically impossible to directly replace these coenzymes with artificial redox compounds like 02 mentioned above. However, if a redox compound is allowed to coexist with the coenzyme, the substrate concentration can be measured by electrochemical measurement of the redox compound. For example, when lactate dehydrogenase is used as the enzyme, NAD is used as the coenzyme, and phenazine methosulfate is used as the redox compound, the dehydrogenation reaction of lactic acid occurs as follows. Lactic acid + phenazine methosulfate (oxidized) lactate dehydrogenase In this case, phenazine methosulfate acts as an electron carrier that accepts electrons via NAD.

The concentration of reduced phenazine methosulfate produced here can be electrochemically measured to determine the lactic acid concentration. As mentioned above, there are various attempts to relate enzymatic reactions to electrochemical reactions.

Among these, conventional examples of enzyme electrodes using immobilized oxidoreductases will be described below. US Patent No. 3
, 542, 662, the outside of the oxygen-permeable membrane of a boularographic oxygen electrode is coated with GO in an acrylamide gel.
An enzyme electrode covered with an immobilized enzyme membrane containing X is shown.

This is a method of electrochemically measuring the concentration of 02 that reacts with glucose using the previously described enzyme as a catalyst. In this method, the measured value of the 02 concentration is greatly influenced by the dissolved oxygen concentration near the electrode, so it is difficult to obtain a stable measured value. In addition, oxygen needs to pass through a double membrane of an enzyme-immobilized membrane and an oxygen-permeable membrane to the electrode, which tends to slow down the response speed. U.S. Patent No. 3,838,03
Specification No. 3 describes an enzyme electrode having a structure in which the outside of the current collector is covered with a semipermeable membrane, and an enzyme (GOX) and a redox compound are present in the space between the semipermeable membrane and the current collector. .

In this case, the redox compound exists in this space in large excess, with some of it remaining undissolved. This is a method of electrochemically measuring the concentration of the redox compound described above. Unlike the oxygen concentration described above, this redox compound concentration maintains a constant value, and a somewhat stable measurement value can be obtained.

However, the semipermeable membrane used here does not allow the passage of enzymes, which are macromolecules, but allows the substrate, which is a small molecule, to freely pass through. Naturally, it moves outside the membrane and is lost. This loss is partially replaced by the dissolution of redox compounds present in undissolved form within the membrane. However, this also has a limit; the electrode life is determined by the loss of redox compounds rather than by the lifespan of the enzyme, and is very short.
Furthermore, even if a microelectrode with this configuration is manufactured and an attempt is made to directly measure the substrate concentration in the living body, it is practically impossible, considering the influence of the elution of the redox compound into the living body. There is also an example of measuring the amount of lactic acid and ethanol as substrates using an enzyme electrode in which a collagen membrane in which lactate dehydrogenase or alcohol dehydrogenase is included and immobilized is closely attached to a current collector.

(BulletinOftheChem.SOc.Of
Japan, ↓Ran (11), 3246, 1975).
In this case, the necessary coenzyme (NAD) and redox compound (phenazine methosulfate) are dissolved in a solution containing the substrate.

Therefore, these expensive reagents are discarded each time a measurement is performed, which is not practical. Furthermore, during measurement, it is necessary to always add a certain amount of redox compounds and coenzymes to the solution, making the measurement operation complicated. Furthermore, direct measurement of living organisms is naturally impossible. As described above, all conventional enzyme electrodes using immobilized oxidoreductases have essential problems, and at present they have not been widely put into practical use. In order to solve these problems,
Attempts have been made to use polymer compounds (generally referred to as redox polymers or redox resins) in which a redox compound is introduced into a polymer.

For example, GOX is used as the enzyme, and a condensation polymer of quinone, formaldehyde, and piperazine hydrochloride represented by the following structural formula is used as the redotus polymer. Figure 1 shows the structure of this enzyme electrode. 1 is a cylindrical electrode support made of an insulating material, 2 is a disk-shaped platinum current collector attached to a recess provided on the bottom surface, and 3 is a platinum current collector. a semipermeable membrane such as cellophane or collodion that covers the support from the bottom to the sides; 4 is a fixing ring for fixing the membrane 3 to the support;
5 is a lead wire connected to the current collector 2.

A space 6 is formed between the current collector and the semipermeable membrane on the bottom surface of the support 1, and the enzyme and redox compound are confined in this space.

That is, since the above redox polymer used in this example is water-soluble, GO
It is dissolved together with X and confined in the space 6 by the membrane 3. In this case, since both the enzyme and the redox polymer are macromolecules, they do not move outside the membrane 3.
This means that it is substantially fixed near the current collector 2. The major difference between this enzyme electrode and the example of U.S. Patent No. 3,838,033 mentioned above is that the redox compound is polymerized, so that the redox compound does not elute out of the electrode system. Not at all.

However, a semi-permeable membrane is required, and the presence of the semi-permeable membrane retards the diffusion of the substrate, resulting in a decrease in response characteristics and does not sufficiently withstand repeated use. The present invention eliminates the above-mentioned disadvantages and provides an enzyme electrode that does not require a semipermeable membrane.

That is, the present invention is characterized in that an oxidoreductase and a redox compound serving as its electron carrier are immobilized integrally with an electron current collector. The present invention will be explained below using examples.

Example 1 As a GOX redox polymer cross-linked with glutaraldehyde as an enzyme, a compound represented by the following structural formula in which thionine was bonded to methylolated polyacrylamide was used, and these were mixed with carbon powder, and the binder body was A base resin powder was added, press-molded, and brought into contact with a platinum current collector.

In the enzyme electrode constructed in this way, the enzyme and the redox compound do not elute into the liquid even without a semipermeable membrane, and the enzyme and the redox compound are integrated with the current collector (carbon), and the second current collector (platinum) ) is fixed.

FIG. 2 shows a measurement system using an enzyme electrode 7, in which 8 is a saturated calomel electrode, 9 is a salt bridge, 10 is a counter electrode, and 11 is a buffer solution containing a substrate.

The above enzyme electrode was set at a constant potential of 0 V with respect to the saturated calomel electrode, and the concentration of glucose, the substrate, was varied from 0 to 3 × 10
When we measure the amount of change in anode current (the oxidation current of the reduced form of redotus polymer) when changing the amount by -3 mol/l, we find that it is approximately 1 for 3 x 10-3 mol/l glucose.
The current increased by 5 μA and reached a steady value in about 1 minute.

Example 2 As a redox polymer, a compound represented by the following structural formula in which quinone was bonded to a methylolated product of a copolymer of styrene and acrylamide was used.

- 8i▼ 8i1 `` 8i▼ 8▼▼ Ii A tetrahydrofuran solution of this compound was applied to the surface of a platinum current collector and dried, and then GOX was directly immobilized thereon using glutaraldehyde to prepare an enzyme electrode. This redox polymer is soluble in tetrahydrofuran but insoluble in buffer solution.In this way, the enzyme and redox compound are integrated and immobilized on the surface of the current collector, and retention by the semipermeable membrane is the same as in Example 1. This is no longer necessary.The structure of this enzyme electrode is shown in Figure 3. 12 is a platinum current collector;
3 is a redox polymer coated on the current collector; 14 is a cross-linked enzyme; and 15 is a lead wire of the current collector.

The characteristics of this electrode showed a current increase of about 40 μA for 3×10 −3 mol/l glucose, and the current reached a steady value in about 1 minute.

The glucose concentration could be measured for about 2 months. Example 3 ArI as a redox polymer
Using a cation exchange resin sold under the name of lberllte IR-120 in which potassium ferricyanide has been absorbed, carbon powder, GOX crosslinked with glutaraldehyde, and fluororesin powder are mixed, and platinum An enzyme electrode was prepared by press molding on a current collector.

When looking at the response to glucose while maintaining a constant potential of 0.4 with respect to a saturated calomel electrode, a current increase of about 15 μA was observed for 3×10 −3 mol/l of glucose, and the current reached a steady value in about 1 minute. did. Similar results were also obtained when an iron salt of polyacrylic acid represented by the following structural formula was used as the redox polymer.

r-6-j? ? - Next, we will explain an example in which a redox compound is directly immobilized on a current collector using chemical bonds.

Example 4 Gallocyanine as a redox compound was directly immobilized on a SnO2 Nesaglass current collector using its surface hydroxyl groups through ester bonds, and GO was further immobilized thereon.
An enzyme electrode was prepared by immobilizing X with glutaraldehyde.

In this SnO2 electrode in which enzymes and redox compounds are integrally immobilized, glucose is 3 x 10-3 mol/! A current increase of 10 μA occurred, and the current reached a steady value in about 2 minutes. Example 5 A carboxyl group on the surface of a carbon current collector was reacted with an isodophenolic acid group to fix a redox compound on the surface of the current collector through an ester bond, and then glutaraldehyde was directly applied to the surface using aldehyde oxidase as an enzyme. cross-linking and immobilization using

The enzyme electrode prepared in this way was made of acetaldehyde 1
×10-3 mol/! A current increase of 5 μA was observed, and the current reached a steady value in about 2 minutes. Next, an enzyme electrode in which not only enzymes and redox compounds are immobilized but also coenzymes is immobilized will be explained.

Example 6 Nicotinamide adenine dinucleotide (NAD), a typical coenzyme, is used and immobilized by covalent bonding to Sepharose, a polysaccharide polymer carrier.

On the other hand, as a redox polymer, a compound represented by the following structural formula in which riboflavin-5'-phosphate is bonded to methylolated polyacrylamide is used. The above-described immobilized NAD and redox polymer were mixed with graphite powder and press-molded, and alcohol dehydrogenase was cross-linked and immobilized on the surface of this molded body using glutaraldehyde.

The electrode on which the enzyme, redox compound, and coenzyme were immobilized in this way was set at a constant potential of -0.1 with respect to the saturated calomel electrode, and the response to ethanol was observed as follows:
The current increased by about 20 ttA for an increase in ethanol concentration of ×10 −3 mol/l, and the current reached a steady value in about 3 minutes.

Example 7 A phosphate buffer solution of 7.7 in which coenzyme NAD and lactate dehydratase were dissolved was added to the mixed powder of graphite powder and the redox polymer used in Example 6, and after drying,
Coenzymes and enzymes are simultaneously cross-linked and immobilized on the mixed powder of graphite and redox polymer by the action of glutaraldehyde.

By press-molding the powder thus produced, an enzyme electrode in which the enzyme, redox compound, and coenzyme are immobilized integrally with the current collector can be obtained. This enzyme electrode is Example 6
Under the same conditions as above, a current increase of about 15 μA was shown for lactic acid of 1×10 −3 mol/′. In the embodiments described above, platinum, carbon (including graphite), and tin oxide were used as current collector materials, but other noble metals such as gold, silver, iridium, and palladium, alloys of these, titanium, and tantalum may also be used. Corrosion-resistant metals and alloys such as, conductive oxides such as ruthenium oxide, and semiconductors such as silicon, germanium, and titanium oxide may also be used.

Furthermore, although a crosslinking method using glutaraldehyde has been described as an enzyme immobilization method, it is also possible to directly bind the enzyme onto a water-insoluble carrier through a covalent bond.

Furthermore, although we have given some examples of redox polymers, these may not necessarily have the exact structure shown in the chemical formula as a polymer. There is also a possibility that it may fall off. In any case, the gist of the present invention is to immobilize a redox compound, which is an electron carrier of an oxidoreductase, and a coenzyme required by the enzyme, together with the enzyme, into a current collector; The types of redox compound derivatives, redox polymers, and surface modifications of current collectors used for this immobilization, as well as the types of immobilization methods for enzymes and coenzymes, are not limited to those in the examples. Furthermore, there may be two or more types of enzymes, redox compounds, and coenzymes, and these can be immobilized by a combination of various immobilization methods instead of a single immobilization method.

Furthermore, regarding enzymes, a complex enzyme that immobilizes an enzyme that does not belong to oxidoreductases together with a oxidoreductase, and in the first step, reacts the substrate of the former in an enzymatic reaction that belongs to the former and converts it into the substrate of the enzyme that belongs to the latter. It is also possible to use a reaction system.

As for enzymes, instead of using natural extracts as they are, they can be used with slight chemical modifications to increase their activity. Furthermore, it is also possible to directly immobilize microorganisms and organelles containing these enzymes without isolating the enzymes from them, and incorporate them into electrodes. As mentioned above,
By integrally immobilizing oxidoreductases, redox compounds, and coenzymes with a current collector, enzymes, redox compounds, and coenzymes can be reused without eluting into the sample liquid even without a semipermeable membrane. It is possible to obtain a highly reliable enzyme electrode that has a long life and is easy to handle.

Furthermore, by creating a micro-composite electrode that compactly combines the measurement electrode system of enzyme electrode, reference electrode, and counter electrode,
It is also possible to directly measure the substrate concentration in a living body in a short time without mixing foreign substances into the biological fluid. Furthermore, the enzyme electrode according to the present invention can be used not only for measuring substrate concentration but also as an electrode for batteries.

For example, a glucose oxidase or thionine immobilized enzyme electrode can constitute a fuel cell using glucose as fuel when an oxygen electrode is used as a counter electrode. Figure 4 shows the structure of the fuel cell. In the figure, 16 is an enzyme electrode, 17 is an oxygen electrode, 18 is a separator, and 19 is an electrolyte, such as a phosphate buffer.

20 is a gas chamber to which oxygen or air is supplied.

A liquid chamber 21 is supplied with a solution of glucose, which is a fuel.

This battery generates a voltage of about 0.7 and can obtain a current value of about 1 mA.

In this case, the enzymes and redox compounds are of course immobilized, and there is no need to add them to the fuel liquid for replenishment, making it a true refueling type battery with a long life of approximately 6 months. In addition, this electrode can also be used to synthesize various substances, such as gluconic acid from glucose (a directly produced gluconolactone hydrolyzate), pyruvic acid from lactic acid, etc. In this case, Another advantage is that the product is not contaminated with foreign substances such as enzymes, redox compounds, and coenzymes, making it extremely easy to isolate the product.

[Brief explanation of the drawing]

Figure 1 is a sectional view of the main parts showing the structure of a conventional enzyme electrode, Figure 2 is a diagram showing the configuration of a measurement system using the enzyme electrode, and Figure 3 is an enzyme electrode according to an embodiment of the present invention. The schematic diagram, FIG. 4, is a schematic diagram of a battery using an enzyme electrode.

Claims (1)

[Claims] 1. A method comprising at least an oxidoreductase, a redox compound serving as an electron carrier for the enzyme, and an electron current collector, and the enzyme and the redox compound are integrally immobilized with the current collector. An enzyme electrode characterized by: 2. The enzyme electrode according to claim 1, wherein the redox compound is a polymer. 3. The enzyme electrode according to claim 1, wherein the redox compound is fixed to the current collector through a chemical bond. 4 At least an oxidoreductase and a coenzyme of this enzyme,
An enzyme electrode comprising a redox compound serving as an electron carrier of the oxidoreductase and an electron current collector, the enzyme, coenzyme, and redox compound being integrally immobilized with the current collector. 5. The enzyme electrode according to claim 4, wherein the redox compound is a polymer. 6. The enzyme electrode according to claim 4, wherein the redox compound is fixed to the current collector through a chemical bond.